ABSTRACT Redbud Labs proposes to develop a low-cost, integrated microfluidic system to purify and concentrate pathogens directly from 10 mL of whole blood, with the goal of improving the sensitivity of downstream blood culture and molecular diagnostic assays used in sepsis diagnosis and treatment. Phase I studies have demonstrated that our technology, based on actuated surface attached posts (ASAPs), can achieve high binding efficiency from a complex matrix, such as whole blood, without fouling. Our goal for this Phase II project is to develop a working prototype of the pathogen purification system, validate the system using clinical blood samples, and prepare for scale-up. The specific aims of this proposal are to: (1) develop an integrated pathogen purification system, (2) validate the system using clinical samples, and (3) optimize ASAP fabrication methods to support scalable production. Prior to commencing the work in Phase II, we will complete an Aim 0 which focuses on evaluating the binding chemistry efficiency of the capture chamber, using the broad-spectrum MBL antibody (that can bind >90% of pathogens that infect blood) in place of the E.coli-specific antibody used in Phase I studies. The integrated system in Phase II will be validated using opportunistic clinical blood samples that are routinely collected from patients admitted to the ICU at Duke University Hospital. Additionally, process development, such as wafer-level surface functionalization, ASAP packaging for integration with a cartridge format, and shelf stability studies will be conducted to ensure expected product performance by the time it reaches the end user. The outcome of this project will be an integrated, inexpensive, and simple system that could transform the current status of pathogen identification and drug susceptibility testing, particularly for rare and low number pathogens in blood. The broader impact of this system is the simplification of capture, recovery and concentration of any analyte from whole blood which could improve the speed and sensitivity of in vitro diagnostics.